The role of chromosomal proteins in maintaining the structure and regulating the function of chromatin is studied. Chromatin segments containing specific nuclear proteins have been isolated by immunoaffinity chromatography. The histones associated with polynucleosomes enriched in the protein, HMG-17, are hyperacetylated. The epitopes of monoclonal antibodies against histone H5 have been mapped. These monoclonal are used to study chromatin rearrangements and to isolate chromatin regions enriched in protein H1. A survey of cells derived from higher and lower eukaryotes revealed that H1-like proteins are present only in higher eukaryotes. A plasmid-containing potential Z-DNA forming sequence has been used to study the binding of the chromosomal proteins, HMG-1 and HMG-2, to DNA in various conformations. The results indicate that HMG proteins can distinguish between various DNA structures and that they bind selectively to distinguishable single-stranded regions. The ability of HMG proteins to distinguish between various DNA structures may have functional implications in gene regulation.